Molecular cloning, characterization and functional analysis of a heat shock protein 70 gene in Cyclina sinensis

Abstract

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily and is involved in protecting organisms against various stressors. In the present study, we used RACE to clone a full-length Cyclina sinensis HSP70 cDNA termed CsHSP70. The full length of the CsHSP70 cDNA was 2308 bp, with a 5′ untranslated region (UTR) of 42 bp, a 3′ UTR of 268 bp, and an open reading frame (ORF) of 1998 bp encoding a polypeptide of 655 amino acids with an estimated molecular mass of 72.75 kDa and an estimated isoelectric point of 5.48. Quantitative real-time PCR was employed to analyze the tissue distribution and temporal expression of the CsHSP70 gene after bacterial challenge and cadmium (Cd) exposure. The CsHSP70 mRNA transcript was expressed ubiquitously in five examined tissues, with the highest expression in hemocytes (P < 0.05) and with the lowest expression in the hepatopancreas. Furthermore, the expression level of CsHSP70 in hemocytes at 3 h after Vibrio anguillarum challenge was extremely significantly up-regulated (P < 0.01). Moreover, the CsHSP70 transcript was up-regulated significantly following exposure to a safe Cd concentration (0.1 mg/L). Finally, after the CsHSP70 gene was silenced by RNA interference, the expression of the CsTLR13 and CsMyD88 genes were extremely significantly decreased (P < 0.01). The results indicated that CsHSP70 could play an important role in mediating the environmental stress and immune responses, and regulating TLR signaling pathway in C. sinensis.